Use of 5-substituted nucleosides and/or prodrugs thereof in the resistance-free treatment of infectious diseases

ABSTRACT

The present invention relates to the use of 5-substituted nucleosides and/or the prodrugs thereof together with at least one active substance in order to produce a drug or combination preparation for resistance-free therapy of infectious diseases caused by bacteria or protozoa.

DESCRIPTION OF BACKGROUND INFORMATION

Resistance development relative to treatment with medicines represents auniversal defense mechanism of microorganisms, animals and plants. Inhumans, this defence mechanism is found in tumors. Generally,multiplication (amplification) of specific genes leads to thedevelopment of resistance. This gene amplification causes theoverproduction of a gene product that directly or indirectly reduces theeffect of the drug. Resistance development of Plasmodia (pathogens ofmalaria), and Leishmania (flagellates as pathogens of cutaneousLeishmaniose i.a.) is based partly on the amplification of the samegenes as in human tumors.

Proof that gene amplification in bacteria leads to resistances has beensuccessful with Proteus bacteria, Escherichia coli, Streptococca andStaphylococca. All are important hospitalism germs and pathogens ofurinary tract infections (Romero and Palacios, gene amplification andgenome plasticity in procaryotes, Ann. Rev. Genet. 1997). The mechanismunderlying gene amplification, recombination, can likewise lead to thedevelopment of resistance. This form of resistance development has beenproved unequivocally without exception in all types of bacteria but alsofor human tumors and all organisms which have been examined to date.

Malaria is a collective term for infections by protozoa of thePlasmodium type. Due to increasing resistance of Plasmodia tochemotherapeutics and of Anopheles mosquitoes to insecticides, thesituation is increasingly deteriorating. Both resistances can be causedby gene amplification/recombination.

4-aminoquinolines (Chloroquine, Amodiaquine, mepacrine and Sontaquineare used as antimalaria drugs. These are analogues of quinine.Furthermore, there are two groups of antifolates, on the one handdihydrofolate reductase (DHFR) inhibitors (pyrimethamine andchloroguanide) and on the other hand the sulphones and sulphonamides.Resistances can therefore arise due to amplification of the DHFR gene(Cowman and Lew, 1989; Cowman and Lew, 1990; Tanaka et al., 1990a;Tanaka et al. 1990b; Watanabe and Inselburg, 1994). The amplification of“Multi-drug-resistance” genes is likewise of importance (Foote et al.,1989). The amplification of the pfmdr1 gene is connected for example toresistance to mefloquine, halofantrine and quinine (Wilson et al., 1989;Cowman et al., 1994). As a result of increasing resistance of thePlasmodia to chemotherapeutics and Anopheles mosquitoes to insecticides,the situation is increasingly deteriorating. Both resistances can becaused by gene amplification/recombination. The development ofresistance of Plasmodia is intended to be prevented by the prevention ofchemotherapy-induced gene amplification.

Leishmaniases are caused by Leishmania (intracellular parasitic protozoaof the Mastigophora class) and infectious diseases transmitted byPhlebotoma (sand mosquitoes). Resistances to a chemotherapy are based onthe amplification of some genes which also play a role inchemoresistance of tumours (Ouellette and Borst, 1991; Grondin et al.,1998; Arana et al., 1998; Heimeur and Ouellette, 1998; Kundig et al.,1999). The development of resistance is intended to be prevented bypreventing chemotherapy-induced gene amplification/recombination.

The use of 5-substituted nucleosides for inhibiting resistancedevelopment during cytostatic treatment is already known from DE 195 45892.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to make possible aresistance-free treatment of infectious diseases caused by bacteria orprotozoa.

In one aspect, the present invention is directed to a method for theresistance-free therapeutic treatment of infectious disease caused bybacteria or protozoa, comprising administering to an individual in needthereof at least one of a 5-substituted nucleoside and a prodrug thereoftogether with at least one active substance.

The at least one of a 5-substituted nucleoside and prodrug thereof andthe active substance can be present in a single formulation.

The at least one of a 5-substituted nucleoside and prodrug thereof andthe active substance can be present in separate formulations.

(E)-5-(2-bromovinyl)-2-deoxyuridines (BVDU), salts thereof, protectiveforms thereof and prod rugs thereof can be used.

The at least one of a 5-substituted nucleoside and a prodrug thereof cancomprise a prodrug of Formula (I).

The at least one active substance can comprise at least one antibiotic.

The bacteria can be Proteus bacteria, Escherichia coli, Streptococca orStaphylococca.

The infectious disease can be caused by Plasmodia, and the at least oneactive substance can comprise at least one of antibiotics andanti-infective agents active against Plasmodia.

The infectious disease can be malaria, and the at least one activesubstance can comprise 4-aminoquinolines.

The at least one active substance can comprise quinine derivatives, andthe quinine derivatives can be Chioroquine, Amodiaquine, mepacrine orSontaquine.

The at least one active substance can comprise antifolates. Theantifolates can be pyrimethamine, chloroguanide, sulphones orsulphonamides.

The infectious disease can comprise Leishmaniases, and the at least oneactive substance can comprise at least one of chemotherapeutics,antibiotics and anti-infectives.

The at least one active substance can comprise at least onechemotherapeutic, and the chemotherapeutic can comprise methotrexate.

The at least one of a 5-substituted nucleoside and a prodrug thereof canbe used in concentrations such that a blood concentration of 0.01 to 10μg/ml results.

The at least one of a 5-substituted nucleoside and a prodrug thereof canbe used in concentrations such that a blood concentration of 0.05 to 5μg/ml results.

The at least one active substance can be used in a therapeuticconcentration.

The administering can comprise administration by at least one ofinjection, orally, rectally, intravaginally, intranasally and localapplication.

The method can further comprise administering additives comprising atleast one of aqueous and non-aqueous solvents, stabilizers, suspensionagents, dispersion agents and wetting agents.

The method can further comprise administering additives comprising atleast one of polyethylene glycols, colourants and perfume agents.

The at least one of a 5-substituted nucleoside and a prodrug thereof andthe at least one active substance can be in the form of a fine powder,powder, suspension, solution, emulsion, salve or paste.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described in the detailed descriptionwhich follows, in reference to the noted drawing by way of anon-limiting exemplary embodiment of the present invention, and wherein:

FIGS. 1 and 2 show the results of growth tests using Kanamycin and BVDU;

FIG. 3 shows comparison of MIC of Kanamycin;

FIG. 4 shows comparison of MIC of Kanamycin;

FIG. 5 shows the results of growth tests with Amikacin and BVDU;

FIG. 6 shows comparison of MIC of Amikacin;

FIG. 7 shows determination of MIC of Kanamycin;

FIG. 8 shows comparison of MIC of Amikacin;

FIG. 9 shows the results of growth tests with methotrexate and BVDU; and

FIG. 10 shows BVDU alone.

DETAILED DESCRIPTION OF THE INVENTION AND EXAMPLES

Resistance-free therapy of infectious diseases from the group ofinfections caused by bacteria or by protozoa is achieved in that,together with the substance which is active against the infectiousdisease, 5-substituted nucleosides and/or the prodrugs thereof areadministered.

The active substance and the 5-substituted nucleosides or the prodrugsthereof can thereby occur both in a single formulation and in separateformulations as a combination preparation. A simultaneous, separate ortemporally separated application can thereby occur.

Preferably, the 5-substituted nucleosides are selected from(E)-5-(2-bromovinyl)-2-deoxyuridines (BVDU), the salts thereof, theprotective forms thereof and the prodrugs thereof. The compound offormula 1 can thereby be used preferably as prodrug.

Antibiotics are used in a preferred embodiment as active substances forresistance-free therapy of bacterial infectious diseases. All bacteriaindiscriminately are thereby possible as the bacteria triggeringinfection because of the proved importance of recombination inresistance development and because of the proved importance of geneamplification, e.g. Proteus bacteria, Escherichia coli, Streptococca andStaphylococca.

For the resistance-free therapy of malaria or of other infections causedby Plasmodia, antibiotics and/or anti-infectives, such as e.g.4-aminoquinolines, are used as active substances. Chloroquine,Amodiaquine, mepacrine and Sontaquine are used particularly preferablythereby.

Antifolates are used as further preferred active substance againstmalaria or other infections caused by Plasmodia. For example,pyrimethamine, chloroguadine, sulphones and sulphonamides are includedhere.

For the resistance-free therapy of Leishmaniases, chemotherapeutics,antibiotics and/or anti-infectives are used preferably as activesubstances. Methotrexate is thereby used as preferred chemotherapeutic.

The 5-substituted nucleosides or the prodrugs thereof are usedpreferably in such concentrations that, after administration, aconcentration of 5-substituted nucleosides or the prodrugs thereof inthe blood of between 0.01 and 10 μg/ml, particularly preferred between0.05 and 5 μg/ml, results.

The active substances are administered in standard concentrationsaccording to the type of infectious disease, as are listed for examplein current drug lists. Reference is made here in particular to the RedList 2001 (Red List Service GmbH, Frankfurt/Main).

Administration both of the active substances and also the 5-substitutednucleosides can be effected by injection, orally, rectally,intravaginally, intranasally and/or by local application.

In addition to the active substance and the 5-substituted nucleosides,there can also be used as further additives, aqueous and non-aqueoussolvents, stabilizers, suspension agents, dispersion agents and wettingagents in the formulations. There are possible as additional additives,e.g. polyethylene glycols, colourants and perfume agents. Theformulation can thereby be effected in the form of a fine powder, apowder, a suspension, a solution, an emulsion, a salve or a paste.

The subject according to the invention is now intended to be explainedin more detail with reference to the following examples and FIGS. 1 to10 without restricting the subject to these examples.

1. EXAMPLE Prevention of Resistance Development in Bacteria

Escherichia coli J53 was used as Gram-negative model organism in orderto test the effectiveness of BVDU. This organism carries the naturallyoccurring plasmid RP4, on which resistances to Ampicillin, Tetracyclineand Kanamycin are encoded.

1.1 Adaptation to Kanamycin

Kanamycin is a naturally occurring aminoglycoside antibiotic. It is usedfor the treatment of bacterial infections, if it is not possible to usePenicillin or less toxic antibiotics. Fields of use are inter alia,infections of the bones, the respiratory tracts and of the skin and alsocomplicated urinary tract infections and endocarditis.

In growth tests with Kanamycin (64 μg/ml), a slow adaptation, i.e.resistance development, of E. coli J53 (RP4) to this concentration ofthe antibiotic was achieved. In the presence of BVDU (1 μg/ml), aninhibition of this resistance development/adaptation occurred. Theresults are illustrated in FIGS. 1 and 2.

The gradual adaptation to 64 μg/ml Kanamycin led to a stable change inthe resistance spectrum relative to the antibiotic which was used.

In order to determine the minimal inhibitory concentrations (MIC),precultures were used initially for which antibiotic-free culture mediumhad been inoculated directly from frozen glycerine cultures. The strain,which had become resistant to 64 μg/ml Kanamycin had with 128 μg/ml afour times increased MIC relative to the starter strain (FIG. 3). Thestrain grown in the presence of 1 μg/ml BVDU, which had achieved noresistance to 64 μg/ml Kanamycin, showed growth inhibition already from16 μg/ml Kanamycin. However even higher antibiotic concentrations stillled to the growth of the inoculum. However, the growth inhibitionprevented a larger cell density in the bacteria culture being reached.

In order to test the stability of the specific resistance features ofthe E. coli strains, the MIC determination was repeated. The alreadytested precultures were again absorbed for this purpose inantibiotic-free culture medium and tested for their Kanamycinresistance. The resistance spectra of the starter strain and of thestrain adapted to 64 μg/ml remained unchanged. However, differences wererevealed in the strain which was pretreated with 64 μg/ml Kanamycin +1μg/ml BVDU. The growth of the inoculum was hereby completely preventedfrom a concentration of 32 μg/ml Kanamycin. The MIC of this straincorresponded hence to that of the starter strain. The results areillustrated in FIG. 4.

This means in summary that BVDU prevents the development of resistanceto antibiotics and increases the sensitivity to antibiotics in aspecific concentration range.

1.2 Adaptation to Amikacin

Amikacin acts against pathogens which are resistant to the remainingaminoglycosides. It is given in the case of severe infectious diseasesof the kidneys, urinary and sexual organs and in infections of therespiratory tracts and of the gastro-intestinal tract.

In growth tests with Amikacin (0.25 μg/ml), a slow adaptation, i.e.resistance development, of E coli J53 (RP4) to this concentration of theantibiotic was achieved. In the presence of BVDU (2 μg/ml), inhibitionof this adaptation occurred (FIG. 5).

The gradual adaptation to 0.25 μg/ml Amikacin led to a stable change inthe resistance spectrum relative to the antibiotic which was used.

The strain which had become resistant to 0.25 μg/ml Amikacin had with 2μg/ml an eight times increased minimal inhibitory concentration (MIC)relative to the starter strain. The strain grown in the presence of 2μg/ml BVDU which had achieved no resistance to 0.25 μg/ml Amikacin,showed growth inhibition already from 0.125 μg/ml Amikacin. Thisinhibition also prevented a larger cell density in the bacteria culturebeing reached with higher antibiotic concentrations. The results can bededuced from FIG. 6.

2. EXAMPLE BVDU Conditioned Growth Inhibition After Aminoglycoside/BVDUPretreatment

In order to determine the minimal inhibitory concentrations (MIC) ofKanamycin or Amikacin relative to aminoglycoside/BVDU-pretreated E coliJ53 (RP4) strains, standard precultures were used which contained noadditives in the culture medium (recovery phase). In addition, it wastested in comparative experiments to what extent the addition of BVDU tothe antibiotic-free precultures changes the resistant spectrum of thestrains.

The presence of BVDU (1 or 2 μg/ml) in the preculture or/and the smallBVDU quantity (0.04–0.05 μg/ml) present due to transfer into the MICbatches sufficed for sustained restriction of the growth ofaminoglycoside/BVDU-pretreated E. coli strains even in the absence ofthe antibiotic. In comparison therewith, the growth without antibioticin the additive-free preculture corresponded to that of the untreated E.coli strain. The results are illustrated in FIGS. 7 and 8.

This implies in summary that BVDU also acts alone after removing anantibiotic. BVDU without pretreatment (antibiotic+BVDU) results in noeffect and is not toxic.

3. EXAMPLE Prevention of Resistance Development in Protozoa

Prevention of resistance development in Zooflagellates (Leishmania) bythe simultaneous administration of the anti-recombinogenic 5-substitutednucleoside (E)-5-(2-bromovinyl)2′-deoxyuridines (BVDU) withmethotrexate.

Protozoa of the Zooflagellata strain, such as e.g. Trypanosoma aspathogens of sleeping sickness and Leishmania (intracellular parasiticprotozoa of the Mastigophora class) as pathogens of Leishmniases areinfectious. Resistances to chemotherapy are based both in Trypanosoma(Wilson et al., 1991) and Leishmania (Ouellette and Borst, 1991; Grondinet al., 1998; Arana et al., 1998; Haimeur and Ouellette, 1998; Kundig etal., 1999) on the amplification of some genes which also play a role inthe chemoresistance of tumours. The resistance development is intendedto be prevented via the prevention of chemotherapy-inducedrecombination/gene amplification.

Methotrexate (MTX) resistant cells of Leidshmania donovanii wereproduced by gradual increase in the MTX concentrations of 5 to 10, of 10to 50 and of 50 to 100 μm MTX. 5×10⁶ cells/ml respectively were seededin the growth medium. The cells were diluted again to 5×10⁶ cells/ml foreach new batch. The cells were then constantly subjected to the nexthigher MTX concentration when the cell division rate of theMTX-subjected cells had stabilised to the control level. This waspossible after respectively approximately three to four passages. If 1μg/ml BVDU was added simultaneously to the cultures, then the celldivision rate never reached the control level, i.e. the cells developed,in contrast to the cells treated with MTX alone, no resistance to thetreatment. The result of this test can be seen in a simplified versionin FIGS. 9 and 10.

1. A method for the resistance-free therapeutic treatment of infectiousdisease caused by bacteria or protozoa selected from Gram-negativebacteria, Trypanosoma or Leishmania, comprising administering to anindividual in need thereof at least one of(E)-5-(2-bromovinyl)-2-deoxyuridine (BVDU), salts thereof, protectiveforms thereof and prodrugs thereof together with at least one activesubstance selected from antibiotics and antiprotozoics.
 2. The methodaccording to claim 1 wherein the at least one of(E)-5-(2-bromovinyl)-2-deoxyuridine (BVDU), salts thereof, protectiveforms thereof and prodrugs thereof and the at least one active substanceselected from antibiotics and antiprotozoics are present in a singleformulation.
 3. The method according to claim 1 wherein the at least oneof (E)-5-(2-bromovinyl)-2-deoxyuridine (BVDU), salts thereof, protectiveforms thereof and prodrugs thereof and the active substance selectedfrom antibiotics and antiprotozoics are present in separateformulations.
 4. The method according to claim 1 wherein the at leastone of (E)-5-(2-bromovinyl)-2-deoxyuridine

(BVDU), salts thereof, protective forms thereof and prodrugs thereofcomprises a prodrug of Formula (I).
 5. The method according to claim 1wherein the at least one active substance selected from antibiotics andantiprotozoics comprises at least one antibiotic selected fromaminoglycosides, beta lactames, tetracyclines and quinolones.
 6. Themethod according to claim 1 wherein the at least one active substanceselected from antibiotics and antiprotozoics comprises quininederivatives, and the quinine derivatives are Chloroquine, Amodiaquine,mepacrine or Sontaquine.
 7. The method according to claim 1 wherein theat least one active substance further includes antifolates.
 8. Themethod according to claim 7 wherein the antifolates comprisepyrimethamine, chloroguanide, sulphones or sulphonamides.
 9. The methodaccording to claim 1 wherein the infectious disease comprisesLeishmaniases.
 10. The method according to claim 9 wherein the at leastone active substance further includes at least one chemotherapeutic, andthe chemotherapeutic comprises methotrexate.
 11. The method according toclaim 1 wherein the at least one of (E)-5-(2-bromovinyl)-2-deoxyuridine(BVDU), salts thereof, protective forms thereof and prodrugs thereof isused in concentrations such that a blood concentration of 0.01 to 10μg/ml results.
 12. The method according to claim 1 wherein the at leastone of (E)-5-(2-bromovinyl)-2-deoxyuridine (BVDU), salts thereof,protective forms thereof and prodrugs thereof is used in concentrationssuch that a blood concentration of 0.05 to 5 μg/ml results.
 13. Themethod according to claim 1 wherein the at least one active substance isused in a therapeutic concentration.
 14. The method according to claim 1wherein the administering comprises administration by at least one ofinjection, orally, rectally, intravaginally, intranasally and localapplication.
 15. The method according to claim 1 further comprisingadministering additives comprising at least one of aqueous andnon-aqueous solvents, stabilizers, suspension agents, dispersion agentsand wetting agents.
 16. The method according to claim 1 furthercomprising administering additives comprising at least one ofpolyethylene glycols, colourants and perfume agents.
 17. The methodaccording to claim 1 where the at least one of(E)-5-(2-bromovinyl)-2-deoxyuridine (BVDU), salts thereof, protectiveforms thereof and prodrugs thereof and the at least one active substanceare in the form of a fine powder, powder, suspension, solution,emulsion, salve or paste.
 18. The method according to claim 1 whereinthe Gram-negative bacteria comprise Esherichia coli.